The objective of this proposal is to investigate the regulation of bone alkaline phosphatase (AP) by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the osteoblast at the transcriptional, translational, and posttranslational levels in phenotypically- distinct osteoblast-like rat and human osteogenic sarcoma cell lines and in fetal rat calvariae. It is well established that the osteoblast possesses specific, high- affinity receptors for 1,25(OH)2D3 and that the hormone acts to modulate the biosynthetic products of the osteoblast, such as type I collagen, osteocalcin, and alkaline phosphatase, at the level of transcription. Recently, phenotypically-distinct subclones of the osteoblast-like rat osteogenic sarcoma (ROS) 17/2.8 cell line have been isolated. 1,25(OH)2D3 stimulates AP activity and osteocalcin secretion in the G2 subclone (which exhibits low basal AP activity) by a genome-dependent mechanism. In contrast, 1,25(OH)2D3 inhibits alkaline phosphatase activity in the C12 subclone (which exhibits high basal AP activity and undetectable levels of osteocalcin secretion) by genome-independent mechanism. The regulation of AP will be examined systematically in these well-defined ROS clones and selectively in human osteogenic sarcoma (HOS) cells and in fetal rat calvariae by: a) direct enzymatic assay of total AP activity, b) quantitation of bone AP levels by immunoassay, and c) conducting parallel studies of bone AP-mRNA levels. RNA will be isolated from control and 1,25(OH)2D2-treated cultures for use in studies which include: i) in vitro translation in a rabbit reticulocyte lysate, followed by immunoprecipitation of bone AP and characterization of the radiolabelled products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), ii) construction of an expression library in the wavelength gtll vector, followed by screening for AP-producing recombinants and production of AP-specific cDNA probes; and iii) agarose gel electrophoresis, Northern transfer, and hybridization with the 32P-labelled AP-cDNA probe. In addition, posttranslational processing and glycosylation will be examined by pulse-labelling with 35S-methionine, 3H-mannose, or 3H-glucosamine, followed by SDS-PAGE as above. This integrated approach should clarify the mechanisms by which AP is either up- or down-regulated depending on the phenotype of the osteoblast and may contribute to a general understanding of how steroid hormones regulate gene expression in eukaryotic animals.